CN111620322B - Method for reducing graphene oxide by utilizing microbial fermentation broth - Google Patents

Abstract

The invention provides a method for reducing graphene oxide by utilizing microbial fermentation broth, belonging to the field of material synthesis; in the invention, firstly, a genetically engineered escherichia coli for heterologously expressing [ FeFe ] hydrogenase is constructed, sterile fermentation liquor is obtained through treatment and is used for reduction of graphene oxide, so that biological green reduction of the graphene oxide at normal temperature and normal pressure is realized.

Description

Method for reducing graphene oxide by utilizing microbial fermentation broth

Technical Field

The invention belongs to the field of material synthesis, and particularly relates to a method for reducing graphene oxide by utilizing microbial fermentation broth.

Background

Carbon atoms inside graphenesp ² The hybrid orbitals form a hexagonal honeycomb structure with each carbon atom perpendicular to the plane of the layerpzThe track can form a large pi bond (similar to benzene ring) penetrating through the whole layer, has excellent electric conduction, optical and mechanical properties, and has important application prospects in the aspects of materials science, environmental fields, biomedicine, drug delivery and the like.

The main preparation method of the graphene comprises the following steps: mechanical exfoliation, epitaxy, chemical vapor deposition, silicon carbide epitaxy, graphene Oxide (GO) reduction, and the like. The principle of the graphene oxide reduction method is that graphite is oxidized to obtain a dispersed graphene oxide solution, and then the graphene oxide is reduced by using a reducing agent, wherein the common reducing agent comprises hydrazine hydrate, dimethylhydrazine, sodium borohydride, alcohols, phenols and the like. Although the reduced graphene oxide prepared by the chemical reduction method exhibits excellent electrochemical properties, stability and excellent physicochemical properties, and has advantages of high yield and mass production, it has problems of high energy consumption, severe reaction conditions, and environmental pollution. Therefore, the development of a green, environment-friendly and low-cost reduction method for graphene oxide has very important significance.

Currently, many green methods for synthesizing reduced graphene oxide are reported. For example, researchers use environmentally friendly Vc as a reducing agent to synthesize reduced graphene oxide, but the high cost limits the development and application of the reduced graphene oxide; or the extract of natural environment-friendly materials (such as green tea extract) is used as a reducing agent to synthesize the reduced graphene oxide, but the disadvantages are limited raw material sources and complex operation; yong et al developed a method for biologically reducing graphene oxide, which adopts the principle that electrons generated by microbial metabolism are used for reducing graphene oxide, and has the advantages of mild reaction conditions, no toxicity and environmental protection, simple operation, low cost and wide sources compared with the prior green reduction method, but the microorganisms are tightly connected with the reduced graphene oxide, and the products are difficult to separate.

Disclosure of Invention

In order to overcome the defects of a graphene oxide reduction method in the prior art, the invention discloses a method for reducing graphene oxide by utilizing microbial fermentation broth, which comprises the steps of firstly constructing heterologous expression [ FeFe]Hydrogenase (fromClostridium acetobutylicumNBRC 13948), the obtained genetically engineered escherichia coli is treated to obtain sterile fermentation liquor, and the sterile fermentation liquor is used for reduction of graphene oxide, so that biological green reduction of the graphene oxide at normal temperature and normal pressure is realized.

In order to solve the problems, the invention provides a method for reducing graphene oxide by utilizing microbial fermentation broth, which comprises the following specific steps:

(1) Pre-construction of heterologous expression [ FeFe ]]Genetically engineered escherichia coli for hydrogenaseE. coli BL21(DE3)；

(2) The genetically engineered escherichia coli is preparedE. coliBL21 (DE 3) is inoculated into a fermentation culture medium, IPTG is added into the fermentation culture medium to induce and express hydrogenase, and the obtained bacterial liquid is subjected to anaerobic fermentation for a period of time in a constant-temperature shaking box to obtain fermentation bacterial liquid;

(3) Taking a proper amount of fermentation broth obtained in the step (2), performing anaerobic centrifugation to obtain a supernatant, and filtering and sterilizing the supernatant by a 0.22 mu m water system filtering membrane to obtain an aseptic anaerobic fermentation broth;

(4) Under anaerobic conditions, taking a proper amount of fermentation broth obtained in the step (3), adding GO aqueous solution and hydrogen, and carrying out constant-temperature shaking culture to convert GO into rGO.

Further, the gene cluster of [ FeFe ] hydrogenase in the step (1) comprises HydA, hydE, hydF and HydG.

Further, in the step (1), the E.coliE. coliBL21 (DE 3) is obtained by the following method: simultaneously transferring recombinant plasmids pETDuet-1-HydAE and pCDFDuet-1-HydFG through an electrotransformation methodE. coliBL21 (DE 3) was competent and uniformly coated on LB plates containing 40. Mu.g/mL streptomycin and 100. Mu.g/mL ampicillin, cultured at 37 ℃After the cultivation, monoclonal PCR was picked up to verify and preserve the strain.

Furthermore, hydA and HydE in the gene cluster of the recombinant plasmid pETDuet-1-HydAE being [ FeFe ] hydrogenase are obtained by being connected with a vector pETDuet-1.

Further, hydF and HydG in the gene cluster of the recombinant plasmid pCDFDuet-1-HydFG [ FeFe ] hydrogenase were obtained by ligation with the vector pCDFDuet-1.

Further, in step (2), the fermentation medium comprises LB medium, pH buffer, carbon source supplement, electron acceptor, streptomycin and ampicillin.

Further, the pH buffer is any one of MOPS buffer solution, HEPES buffer solution, tris-HCl buffer solution and PBS buffer solution, and the concentration of the pH buffer is 10-100 mmol/L; the carbon source supplement is any one of glucose, sodium lactate, sodium acetate and sodium formate; the electron acceptor is any one of succinic acid, pyruvic acid and carbon dioxide; the streptomycin concentration is 40 mug/mL; the ampicillin concentration was 100. Mu.g/mL.

Further, the OD of the bacterial liquid in the step (2) induced expression ₆₀₀ 0.2 to 2; the final concentration of IPTG is 0.01-1 mmol/L, and the fermentation time is 4-36 h.

Further, in the step (4), the final concentration of GO in the system is 0.1-2 mg/mL, and the final concentration of hydrogen is 0.1-50%.

Further, the constant temperature shaking culture conditions in the step (2) and the step (4) are: the temperature is 4-60 ℃, the rotating speed of the shaking table is 100-300 rpm, and the culture time is 4-36 h.

The invention has the beneficial effects that:

the traditional rGO nano material mainly adopts a chemical reduction method, and the method has high energy consumption and harsh reaction conditions, and is easy to pollute the environment due to the addition of toxic chemical reagents. The rGO microbial fermentation broth reduction method is mild in reaction condition, environment-friendly, free of toxic and harmful chemical reagents, environment-friendly, low in cost, easy to operate, wide in source, easy to separate and purify products and the like compared with other green synthesis methods.

Drawings

FIG. 1 is a schematic representation of the recombinant plasmid described in example 1, wherein a is pETuet-1-HydAE and b is pCDFDuet-1-HydFG.

FIG. 2 is a DNA agarose gel electrophoresis chart of the recombinant plasmid double digestion assay described in example 1, wherein FIG. a is pETDuet-1-HydAE and FIG. b is pCDFDuet-1-HydFG.

FIG. 3 is a macroscopic view of the different bacterial solutions of example 3 before GO is reduced, wherein a is the fermentation broth of the wild strain and b is the fermentation broth of the organism according to the invention.

FIG. 4 is a macroscopic view of the reduced GO from different bacterial solutions of example 3, wherein a is the wild strain broth and b is the biological broth according to the present invention.

Fig. 5 is an XPS characterization graph of rGO prepared in example 4. Wherein a graph a is an XPS graph of GO, b is an XPS graph of rGO, C is a C1s peak-splitting graph in the graph a, and d is a C1s peak-splitting graph in the graph b.

Detailed Description

The present invention will be described in detail with reference to examples below for better understanding of technical spirit of the present invention, but the scope of the present invention is not limited to the following embodiments.

Example 1: construction of heterologous expression [ FeFe ] hydrogenase recombinant plasmid:

(1) Preparation of hydrogenase (HydA) and related mature enzyme (HydE, hydF, hydG):

first, byClostridium acetobutylicumNBRC 13948 genome (Accession number: NC-003030.1) is used as a template, and hydrogenase (HydA) and related mature enzyme (HydE, hydF, hydG) are obtained by high-fidelity PCR amplification. The nucleotide sequence SEQ ID NO of hydrogenase (HydA) and related mature enzyme (HydE, hydF, hydG) is shown in 1-4, and specific conditions of primer sequence, cleavage site, annealing temperature and extension time are shown in Table 1:

TABLE 1 PCR amplification primer sequences and reaction conditions

(2) Constructing a recombinant plasmid pETDuet-1-HydAE:

double digestion of pETDuet-1 plasmid (from Novagen) and HydE gene fragment simultaneously with Nde I and Kpn I sites, obtaining ligation product pET-HydE through cohesive end ligation, transferring the ligation product pET-HydE intoE. coli JM109 was competent (available from Biotechnology (Shanghai) Co., ltd.) and was uniformly coated on 100. Mu.g/mL ampicillin LB plate, and after overnight incubation at 37℃was performed, the monoclonal was selectedE. coli JM109-pET-HydE is cultivated and recombinant plasmid pETDuet-1-HydE is extracted; the pETDuet-1-HydE plasmid and the HydA gene fragment are subjected to double digestion of the Nco I and BamH I sites simultaneously, a connection product pET-HydA E is obtained through viscous end connection, and the connection product pET-HydA E is transferred intoE. coli JM109 was competent, and was uniformly coated on 100. Mu.g/mL ampicillin LB plate, and after overnight incubation at 37℃the monoclonal was picked upE. coli JM109-pET-HydAE was cultured and recombinant plasmid pETDuet-1-HydAE was extracted.

(3) Construction of recombinant plasmid pCDFDuet-1-HydFG:

the pCDFDuet-1 plasmid (derived from Novagen) was digested simultaneously with Nde I and Kpn I sites to obtain a ligation product pCD-HydG by cohesive end ligation, and the ligation product pCD-HydG was transferred intoE. coli JM109 was competent, and was uniformly coated on 100. Mu.g/mL ampicillin LB plate, and after overnight incubation at 37℃the monoclonal was picked upE. coli JM109-pCD-HydG was cultured and recombinant plasmid pCDFDuet-1-HydG was extracted; then, the pCDFDuet-1-HydG plasmid and the HydF gene fragment were digested simultaneously with the Nco I and BamH I sites, and ligated by means of the cohesive ends to give ligation product pCD-HydFG, which was transferred intoE. coli JM109 was competent, and was uniformly coated on 100. Mu.g/mL ampicillin LB plate, and after overnight incubation at 37℃the monoclonal was picked upE. coli JM109-pCD-HydFG was cultured and the recombinant plasmid pCDFDuet-1-HydFG was extracted.

The schematic diagrams of the recombinant plasmids pETDuet-1-HydAE and pCDFDuet-1-HydFG are shown in FIG. 1. From the figure, it can be seen that HydA and HydE gene fragments were inserted into plasmid pETDuet-1 at the Nco I and BamH I sites and Nde I and Kpn I sites, respectively, to construct a new recombinant plasmid pETDuet-1-HydAE. The HydF and HydG gene fragments were inserted into the plasmid pCDFDuet-1 at the Nco I and BamH I sites and the Nde I and Kpn I sites, respectively, to construct a novel recombinant plasmid pCDFDuet-1-HydFG.

(4) Verifying the recombinant plasmid:

extracting pETDuet-1-HydAE and pCDFDuet-1-HydFG recombinant plasmids, carrying out double digestion by Nco I, bamH I or Nde I, kpn I restriction enzymes to obtain a double digestion product, and carrying out agarose gel electrophoresis detection on the double digestion product and HydA, hydE, hydF, hydG target gene fragments. FIG. 2 is a DNA agarose gel electrophoresis chart of the recombinant plasmid double enzyme digestion assay according to the present example, wherein FIG. a is pETDuet-1-HydAE and FIG. b is pCDFDuet-1-HydFG. In FIG. a, band M is the result of electrophoresis of marker, 1 is pETDuet-1-HydE Ndel, kpn I double enzyme digestion, 2 is pETDuet-1 Nde I, kpn I double enzyme digestion, 3 is HydE,4 is pETDuet-1-HydAE Nco I, bamH I double enzyme digestion, 5 is pETDuet-1-HydE Nco I, bamH I double enzyme digestion, 6 is HydA. In FIG. b, band M is the result of marker electrophoresis, 1 is pCDFDuet-1-HydG Ndel, kpn I double enzyme digestion, 2 is pCDFDuet-1 Nde I, kpn I double enzyme digestion, 3 is HydG,4 is pCDFDuet-1-HydFG Nco I, bamH I double enzyme digestion, 5 is pETDuet-1-HydG Nco I, bamH I double enzyme digestion, 6 is HydF.

As can be seen from the figure, the gene size obtained by double digestion of pETDuet-1-HydA by using Nco I and BamH I is 1749bp, and is consistent with the size of gene HydA; the size of the gene obtained by double digestion with Nde I and Kpn I is 1053bp, which is consistent with the size of the gene HydE. The gene size obtained by carrying out double enzyme digestion on pCDFDuet-1-HydFG by adopting Nco I and BamH I is 1235bp, and is consistent with the size of the gene HydF; the gene size obtained after double digestion by Nde I and Kpn I is 1419bp, which is consistent with the size of HydG. Thus, the HydA, hydE gene fragments have been successfully ligated to pETDuet-1, hydF, hydG gene fragments have been successfully ligated to pCDFDuet-1 plasmid, i.e., pETDuet-1-HydAE and pCDFDuet-1-HydFG recombinant plasmids were successfully constructed.

Example 2: heterologous expression of [ FeFe]Hydrogenase enzymeE. coliConstruction of BL21 (DE 3) and determination of expression

(1) The construction method comprises the following steps:

recombinant plasmid pETDueSimultaneous transfer of t-1-HydAE and pCDFDuet-1-HydFGE. coliBL21 (DE 3) competent (available from Biotechnology (Shanghai) Co., ltd.) was uniformly applied to LB plates containing 40. Mu.g/mL streptomycin and 100. Mu.g/mL ampicillin, cultured overnight at 37℃and then the monoclonal was selectedE. coliBL21 (DE 3) -HydAEFG culture.

(2) Heterologous expression of [ FeFe ] hydrogenase:

the recombinant strain was inoculated in 10 mL of LB medium (containing 40. Mu.g/mL of streptomycin and 100. Mu.g/mL of ampicillin) and cultured overnight in a constant temperature shaker at 37℃and 200 rpm. Inoculating the overnight cultured bacterial liquid into 200 mL LB liquid medium (100 mM 3-morpholinopropane sulfonic acid (MOPS) solution, pH is adjusted to 7.2; 40 μg/mL streptomycin and 100 μg/mL ampicillin) at 1% (v/v), placing shake flask into a shaking table at 37deg.C constant temperature of 200 rpm for culturing 1-8 h, and bacteria OD ₆₀₀ Reaching 0.2-2. The bacterial liquid is transferred from the shake flask to a 500 mL anaerobic flask, and 0.01-1 mmol/L IPTG, 20 mM glucose and 25 mM fumaric acid are added. Introducing sterile nitrogen into an anaerobic bottle for more than 30 min for anaerobic treatment, and sealing with a butyl rubber plug and an aluminum cover. The sealed anaerobic bottle is put into a 30 ℃ constant temperature shaking table of 200 rpm for fermentation culture 4-36 h.

(3) [ FeFe ] method for detecting the enzymatic activity of hydrogenase:

[FeFe]the hydrogenase enzyme activity is measured at the rate of hydrogen production under conditions of excess reducing methyl viologen as an electron donor. The specific detection steps are as follows: under anaerobic conditions, preparing a reaction solution containing 100 mM Tris-HCl (pH 7), 150 mM NaCl, 5 mM methyl viologen and 25 mM sodium dithionite, placing the reaction solution in a 10 ml penicillin bottle, and sealing the reaction solution by an aluminum cover and a butyl rubber plug; the headspace gas was nitrogen, and the cell suspension was added to the reaction solution by syringe, 37 ^o The reaction was carried out under the condition C for 4 min, and then 10% (w/v) trichloroacetic acid was added to terminate the reaction. The hydrogen content in the headspace was determined by gas chromatography (GC-7900, tianmei) using a 5A molecular sieve packed column (3 mm x 2 m), a thermal conductivity cell detector (TCD), nitrogen as carrier gas, and a column temperature of 60 ^o C, the temperature of the sample inlet is 120 DEG C ^o C. Detection results such asAs shown in table 2,E. coli BL21 (DE 3) -HydAEFG has the activity of catalyzing hydrogen generation without expressing the wild type hydrogenaseE. Coli BL21 (DE 3) did not have the ability to catalyze the production of hydrogen, demonstrating recombinant strainsE. coliSuccessful heterologous expression of active [ FeFe ] by BL21 (DE 3) -HydAEFG]Hydrogenase.

TABLE 2 results of hydrogenase enzyme activity detection

Example 3: method for reducing graphene oxide by using microbial fermentation broth of heterogenous expression hydrogenase

Taking the fermented bacterial liquid of example 2, taking the supernatant by anaerobic centrifugation, and passing through a reactor under anaerobic conditions of 0.22μAnd filtering and sterilizing by using an m-water system filtering membrane to obtain the sterile anaerobic fermentation liquid. Adding GO with the final concentration of 0.1-2 mg/mL and hydrogen with the final concentration of 0.1-50% into the fermentation broth, carrying out constant-temperature shaking culture under the conditions of 4-50 ℃ and 100-250 rpm of a shaking table, and slowly reducing GO into rGO for 4-36 h. The pre-reaction macroscopic photograph is shown in FIG. 3, with both a and b showing a pale yellow transparent color of GO. The macroscopic photograph after the reaction is shown in fig. 4: the fermentation liquor in the step a is changed from light yellow to yellow brown and no sediment is generated; b the fermentation liquor isE. coliBL21 (DE 3) -HydAEFG broth, liquid changed from pale yellow to black, and black precipitate was clearly seen from the figure. Demonstration of heterologous expression onlyE. coliBL21 (DE 3) -HydEFG broth reduced GO to black rGO due toE. coliThe fermentation liquid of BL21 (DE 3) -HydAEFG exists [ FeFe ]]Hydrogenase oxidizes hydrogen to reduce GO.

Example 4: verification of rGO materials

XPS characterization sample preparation: taking the rGO solution obtained by reducing GO in example 3, centrifuging at 10000 rpm for 10 min, discarding the supernatant, washing 3 times with pure water, washing 3 times with absolute ethyl alcohol, freeze-drying by using a vacuum freeze dryer to obtain a rGO sample, grinding the sample into powder, and performing X-ray photoelectron spectroscopy (XPS) characterization.

As shown in fig. 5, in the case of unchanged carbon element, the peak of rGO was significantly reduced compared to the oxygen element peak of GO in the XPS graph, and the C1s peak-splitting graph shows that most of the oxygen-containing functional groups in rGO have been removed compared to GO, demonstrating that GO has been reduced.

The examples are preferred embodiments of the present invention, but the present invention is not limited to the above-described embodiments, and any obvious modifications, substitutions or variations that can be made by one skilled in the art without departing from the spirit of the present invention are within the scope of the present invention.

Sequence listing

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gcttctgaag ttgaaaaaat acctaacgaa aaggttaaat caatagtaaa aaaacacctt 1380

accgaattaa aagagggaca aagagatttt agattctaa 1419

Claims (7)

1. A method for reducing graphene oxide by using microbial fermentation broth, which is characterized by comprising the following steps:

(1) Pre-construction of heterologous expression [ FeFe ]]Genetically engineered escherichia coli for hydrogenaseE. coli BL21(DE3)；

(2) The genetically engineered escherichia coli is preparedE. coliBL21 (DE 3) is inoculated into a fermentation culture medium, IPTG is added into the fermentation culture medium to induce and express hydrogenase, and the obtained bacterial liquid is subjected to anaerobic fermentation for a period of time in a constant-temperature shaking box to obtain fermentation bacterial liquid; OD600 of the bacterial liquid is 0.2-2 during induction expression; the final concentration of IPTG is 0.01-1 mmol/L; the fermentation time is 4-36 h; the constant temperature shaking culture conditions are as follows: the temperature is 4-60 ℃, and the rotating speed of the shaking table is 100-300 rpm;

(3) Taking a proper amount of fermentation broth obtained in the step (2), performing anaerobic centrifugation to obtain a supernatant, filtering and sterilizing the supernatant, and obtaining a sterile anaerobic fermentation broth;

(4) Under anaerobic conditions, taking a proper amount of fermentation broth obtained in the step (3), adding GO aqueous solution and hydrogen, and carrying out constant-temperature shaking culture to convert GO into rGO; the constant temperature shaking culture conditions are as follows: the temperature is 4-60 ℃, the rotating speed of the shaking table is 100-300 rpm, and the culture time is 4-36 hours; the final concentration of GO in the system is 0.1-2 mg/mL, and the final concentration of hydrogen is 0.1-50%.

2. The method for reducing graphene oxide using a microbial fermentation broth according to claim 1, wherein the gene clusters of the [ FeFe ] hydrogenase in step (1) are HydA, hydE, hydF and HydG.

3. The method for reducing graphene oxide by using a microbial fermentation broth according to claim 1, wherein in the step (1), the escherichia coli e.coli BL21 (DE 3) is obtained by the following method: recombinant plasmids pETDuet-1-HydAE and pCDFDuet-1-HydFG were simultaneously transferred into E.coli BL21 (DE 3) competent by electrotransformation method, and uniformly coated on LB plate containing 40. Mu.g/mL streptomycin and 100. Mu.g/mL ampicillin, after culturing at 37 ℃, monoclonal PCR was selected for verification and strain preservation.

4. The method for reducing graphene oxide by using a microbial fermentation broth according to claim 3, wherein HydA and HydE in a gene cluster of the recombinant plasmid pETDuet-1-HydAE being [ FeFe ] hydrogenase are obtained by connecting with a vector pETDuet-1; the recombinant plasmid pCDFDuet-1-HydFG is obtained by ligating HydF and HydG in the gene cluster of [ FeFe ] hydrogenase with the vector pCDFDuet-1.

5. The method for reducing graphene oxide using a microbial fermentation broth according to claim 1, wherein in step (2), the fermentation medium comprises LB medium, pH buffer, carbon source supplement, electron acceptor, streptomycin, and ampicillin.

6. The method for reducing graphene oxide using a microbial fermentation broth according to claim 5, wherein the pH buffer is any one of MOPS buffer solution, HEPEs buffer solution, tris-HCl buffer solution, and PBS buffer solution; the carbon source supplement is any one of glucose, sodium lactate, sodium acetate and sodium formate; the electron acceptor is any one of succinic acid, pyruvic acid and carbon dioxide.

7. The method for reducing graphene oxide by using a microbial fermentation broth according to claim 5, wherein the final concentration of the pH buffer is 10-100 mmol/L; the final concentration of the carbon source supplement is 10-100 mmol/L; the final concentration of the electron acceptor is 10-100 mmol/L; the final concentration of the streptomycin is 40 mug/mL; the final concentration of ampicillin was 100. Mu.g/mL.